Inhibiting Bcl-2-regulated intestinal fibrosis in models of Crohn’s Disease

纤维化是许多慢性疾病中导致器官衰竭的最后常见途径，在美国每年导致超过1420亿美元的医疗费用。肠纤维化推动了克罗恩病的手术需求，三分之二的克罗恩病患者都需要手术。尽管这对美国的医疗保健和克罗恩病患者产生了巨大的影响，但我们没有治疗肠道纤维化的药物。我们的初步数据表明，靶向Bcl-2信号通路的小分子可以抑制肠成纤维细胞的激活。本提案的目的是验证Bcl-2信号作为肠纤维化的重要途径。该研究项目的长期目标是开发Bcl-2抑制剂，从而为克罗恩病的肠道纤维化提供有效的医学治疗。我们的初步数据表明，候选Bcl-2抑制剂在体外和人类肠道类器官中抑制人肠肌成纤维细胞的激活。然而，这些化合物的效力有限，并且在小鼠全身暴露后可能具有毒性。现有的Bcl-2抑制剂navitoclax已被证明对人类是安全的，目前正处于癌症的2期临床试验中。我们建议对现有化合物进行改进，以提高化合物效力和肠道特异性，以降低全身毒性。在Aim 1中，我们将反复开发和测试这些化合物的体外靶标特异性和抗纤维化活性的新变体。效力将在两个独立的肌成纤维细胞激活模型中显示，使用TGFβ（细胞因子激活）和基质刚度（机械激活）。在Aim 2中，我们将通过评估口服到肠道的吸收以及血浆和肝微粒体中化合物的不稳定性来优化Aim 1中最有前途的Bcl-2抑制剂的药代动力学特性。在Aim 3中，我们将确定有希望的候选化合物的最大耐受剂量，并在小鼠中进行PK和PD测试。有前景的肠道选择性化合物将在鼠伤寒沙门菌小鼠肠道纤维化模型和大鼠三硝基苯磺酸（TNBS）肠道纤维化模型两种啮齿动物模型中逐步测试这些化合物的生物学功效。拟议的研究具有创新性，因为它们将产生抑制新型抗纤维化途径的肠道选择性化合物，并在两种啮齿动物模型中严格测试最佳候选化合物。这项研究计划的重要意义在于，它有可能通过验证器官纤维化的这一途径来影响患者的护理，这是克罗恩病和大多数形式的慢性器官衰竭（包括肾衰竭和肝硬化）的重要临床需求。

Fibrosis is the final common pathway to organ failure in many chronic diseases, leading to over $142 billion in annual medical costs in the United States. Intestinal fibrosis drives the need for surgery in Crohn's disease, which is required in two-thirds of all patients. Despite this immense impact on U.S. healthcare and patients with Crohn's disease, we have no medical therapies for intestinal fibrosis. Our preliminary data show that small molecules targeting the Bcl-2 signaling pathway can inhibit the activation of intestinal fibroblasts. The objective of this proposal is to validate Bcl-2 signaling as an important pathway for intestinal fibrosis. The long-term objective of this research program is to develop inhibitors of Bcl-2 that will lead to effective medical therapies for intestinal fibrosis in Crohn's disease. Our preliminary data demonstrate that candidate Bcl-2 inhibitors inhibit human intestinal myofibroblast activation in vitro and in human intestinal organoids. However, these compounds have limited potency, and potential toxicity from systemic exposure in mice. An existing Bcl-2 inhibitor, navitoclax, has been proven safe in humans and is now in phase 2 clinical trials for cancer. We propose to improve upon the current compounds to enhance compound potency and gut specificity to reduce systemic toxicity. In Aim 1, we will iteratively develop and test novel variants of these compounds for in vitro target specificity and anti-fibrotic activity. Potency will be shown in two independent models of myofibroblast activation, using TGFβ (cytokine activation) and matrix stiffness (mechanical activation). In Aim 2, we will optimize the pharmacokinetic properties of the most promising Bcl-2 inhibitors from Aim 1 by evaluating oral absorption into the gut and compound instability in plasma and liver microsomes. In Aim 3, we will determine the maximum tolerated dose of promising candidate compounds, and perform PK and PD testing in mice. Promising gut-selective compounds will progress to stepwise testing of the biologic efficacy of these compounds in two rodent models, the Salmonella typhimurium mouse model of intestinal fibrosis, and the rat trinitrobenzene sulfonic acid (TNBS) model of intestinal fibrosis. The proposed studies are innovative in that they will generate gut-selective compounds that inhibit a novel antifibrotic pathway, and rigorously test the best candidates in two rodent models. This research proposal is significant in that it has the potential to impact patient care by validating this pathway in organ fibrosis, a significant unmet clinical need in Crohn's disease and in most forms of chronic organ failure, including kidney failure and liver cirrhosis.